Trotta Edoardo, Del Grosso Nicoletta, Erba Maura, Melino Sonia, Cicero Daniel, Paci Maurizio
Istituto di Neurobiologia e Medicina Molecolare, Consiglio Nazionale delle Ricerche, Roma, Italy.
Eur J Biochem. 2003 Dec;270(23):4755-61. doi: 10.1046/j.1432-1033.2003.03877.x.
The structural changes produced by the minor-groove binding ligand DAPI (4',6-diamidine-2-phenylindole) on individual strands of trinucleotide repeat sequences were detected by electrophoretic band-shift analysis and related to their effects on DNA replication in vitro. Among the 20 possible single-stranded trinucleotide repeats, only the T-rich strand of the AAT.ATT triplet exhibits an observable fluorescence band and a change in electrophoretic mobility due to the drug binding. This is attributable to the property of DAPI that favours folding of the random coil ATT strand into a fast-migrating hairpin structure by a minor-groove binding mechanism. Electrophoretic characteristics of AAT, ACT, AGT, ATG and ATC are unchanged by DAPI, suggesting the crucial role of T.T with respect to A.A, C.C and G.G mismatch, in favouring the binding properties and the structural features of the ATT-DAPI complexes. Primer extension experiments, using the Klenow fragment of DNA polymerase I, demonstrate that such a selective structural change at ATT targets presents a marked property to stall DNA replication in vitro in comparison with the complementary AAT and a random GC-rich sequence. The results suggest a novel molecular mechanism of action of the DNA minor-groove binding ligand DAPI.
通过电泳带移分析检测了小沟结合配体DAPI（4',6-二脒基-2-苯基吲哚）对三核苷酸重复序列单链产生的结构变化，并将其与它们对体外DNA复制的影响相关联。在20种可能的单链三核苷酸重复序列中，只有AAT.ATT三联体富含T的链由于药物结合而呈现出可观察到的荧光带和电泳迁移率变化。这归因于DAPI的特性，即通过小沟结合机制有利于将无规卷曲的ATT链折叠成快速迁移的发夹结构。DAPI对AAT、ACT、AGT、ATG和ATC的电泳特性没有影响，表明T.T相对于A.A、C.C和G.G错配的关键作用，有利于ATT-DAPI复合物的结合特性和结构特征。使用DNA聚合酶I的Klenow片段进行的引物延伸实验表明，与互补的AAT和富含GC的随机序列相比，ATT靶点处这种选择性的结构变化在体外具有显著的使DNA复制停滞的特性。结果提示了DNA小沟结合配体DAPI一种新的分子作用机制。
